The World Health Organization (WHO) estimates 500 million clinical cases of malaria and about one million deaths per year. The majority of malaria morbidity and mortality occurs in sub-Saharan Africa. Malaria prevention relies primarily on insecticide-based vector control. The same classes of insecticide used in vector control strategies also are used as pesticides for agricultural purposes. The extensive and multifaceted use of insecticides has led to the emergence and rapid spread of resistance. A key element in resistance management is the identification of resistance mechanisms, which would allow for the development of more reliable resistance monitoring tools and resistance management strategies. The understanding of the molecular mechanisms of insecticide resistance in Anopheles gambiae mosquitoes, the most important malaria vector in sub-Saharan Africa, has progressed in association with technological advancement. In particular, gene amplification-based techniques have allowed the identification of point mutations in coding regions of the para-type sodium channel gene that causes reduced sensitivity of the encoded protein to pyrethroid insecticides. Microarray techniques have taken the analyses of insecticide resistance mechanisms to genome-wide expression profiling. However, microarrays can only determine the relative transcription levels of the genes spotted on the array; genes not represented in the array will be missed, and sequence variation of resistance-related genes will not be examined. High-throughput genome- wide RNA profiling based on the RNA-seq technology is emerging as an improved method for expression studies. The whole transcriptome of mosquitoes is sampled by RNA-seq, and more precise measurement of transcription levels can be obtained. Furthermore, this new technique makes it possible to quantitatively measure known and novel isoforms and detect molecular polymorphisms in resistance-related transcripts. The objective of this application is to use RNA-seq technique to better understand the molecular mechanisms of pyrethroid resistance in An. gambiae mosquitoes. The Specific Aims are: 1) to use RNA-seq technology to determine transcriptome changes and molecular polymorphisms associated with pyrethroid resistance and 2) to use a population genetics approach to determine the role of the resistance candidate genes on resistance phenotypes in field An. gambiae mosquitoes from Kenya. This project will significantly enhance the knowledge of the molecular mechanism of insecticide resistance in An. gambiae, and may lead to the development of more reliable resistance monitoring tools. PUBLIC HEALTH RELEVANCE: Malaria is affecting millions of people, primarily children under the age of five in sub-Saharan Africa. Malaria prevention strategies involve mainly the use of insecticides, and insecticide resistance becomes one of the most important issues in malaria control. The aim of this project is to use the novel RNA-seq technique to better understand resistance mechanisms; the results of this project may lead to the development of more resistance monitoring tools.